Cell Mediated Immune (CMI) responses are commonly used to define the immune status of an individual. Typically, in the art of clinical immunology, the term CMI response encompasses in vivo skin testing, lymphocyte proliferation assays, and the in vitro detection of cytokines produced by peripheral blood mononuclear cells (PBMC) in the presence of a specific antigen. The invention described herein addresses improved methods for manipulating, stabilising and preparing cells derived from isolated whole blood samples for use in assay techniques designed to measure one class of CMI responses, namely the in vitro cytokine-based CMI response to a specific antigen (hereinafter referred to as a “CMI Assay”).
The cells of the immune system are capable of producing immune effector molecules such as cytokines following stimulation by an antigen. CMI Assays involve incubating a cell sample with an antigen and measuring for the presence or quantity of an immune effector molecule such as a cytokine to provide an indication of the ability of the individual to generate a cell-mediated immune response to the selected antigen. Cells for use in a CMI Assay also include isolated populations of lymphocytes (particularly T Cells) and Antigen Presenting Cells (APCs). APCs are involved in processing the antigen in order that the latter may be recognised by T Cell receptors located on the surface of each T Cell. Antigen-induced cytokines may be released into the assay medium and detected directly by, for example, ELISA methods, or quantified in terms of the frequency of cytokine-secreting T Cells using ELISPOT methods.
The filter immunoplaque assay, otherwise called the enzyme-linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody-secreting B cells. At the time it was developed, the technique provided a rapid and versatile alternative to conventional plaque-forming cell assays. Recent modifications have improved the sensitivity of the ELISPOT such that cells producing as few as 100 molecules of a specific protein per second can be detected. These assays take advantage of the relatively high concentration of a given proteinaceous cell product (such as a cytokine) in the environment immediately surrounding the protein-secreting cell. These cell products are captured and detected using high-affinity antibodies. The ELISPOT assay has been reviewed in Current Protocols in Immunology (1994; Pub. John Wiley & Sons, Inc.), Unit 6.19 pages 6.19.1-8.
The ELISPOT assay typically involves six steps: (1) coating a purified cytokine-specific antibody to a membrane-backed microtiter plate; (2) blocking the plate to prevent non-specific absorption of any other proteins; (3) incubating the cytokine-secreting cells with appropriate reagents; (4) removal of cells and reagents; (5) adding a labelled second anti-cytokine antibody; and (6) detecting the antibody-cytokine complex on the membrane.
Methods for isolating subsets of immune cells for analysis using ELISPOT assays have previously been disclosed. See Peters et al., Methods in Molecular Biology, Handbook of ELISPOT: Methods and Protocols (2005), 302, pp. 95-115. Current methods to prepare cells from a whole blood sample for use in a CMI assay involve the isolation of peripheral blood mononuclear cells (PBMCs) using density separation methods such as a Ficoll gradient. In accordance with these methods, and in order to be effective in the CMI assay, lymphocytes and APCs must be purified from the whole blood sample as soon as possible, and in particular within 8 hours of collection of the blood sample from an individual. Janetzski, S. et al., Chapter 4: “Standardisation and Validation Issues of the ELISPOT assay”, in Handbook of ELISPOT (Ed. A. E. Kalyuzhny; Humana Press, New Jersey; 2005), page 80, Note 5. This latter observation was confirmed by Meier et al., who showed that the number of spot-forming T cells as measured in a CMI assay diminished significantly following storage of isolated blood for 1-2 days. Meier et al., Eur. J. Clin. Microbial. Infect. Dis. (2005), 24, pp. 529-536; see Figure 2 in particular.
Several authors have suggested that granulocytes impair T cell function. In 1998, Saxton and Pockley examined the expression of the activation marker CD11b on neutrophils (a type of granulocyte) present in whale blood isolated from healthy laboratory volunteers. Following incubation at Room Temperature (RT) and 4° C., these authors showed that expression of the CD11b antigen by peripheral blood neutrophils was up-regulated after relatively short periods of in vitro incubation. Saxton and Pockley, J. of Immunological Methods (1998) 214:11-17 and Bartels and Schoorl, Clin. Lab. Haem (1998) 20, 165-168 also examined granulocyte activation and degranulation following storage of whole blood isolated from 20 subjects, using CD63 and CD67 granulocyte degranulation markers, and demonstrated an increase in mean CD63 and CD67 antigen expression following blood storage. Bartels and Schoorl also showed that, following whole blood incubation, granulocytes ‘shift’ into the monocyte region of a blood cell scattergram, as defined by the Sysmex NE-8000 haematology analyser used in these studies. Bartels and Schoorl, Clin. Lab. Haem (1998) 20: 165-168.
Other studies have provided evidence suggesting that: (a) when activated, granulocytes can be responsible for inhibiting T cell function; (b) the release of reactive oxygen species during the activation of granulocytes contributes to T cell dysfunction; and (c) a crucial T cell-signalling molecule, namely p56lck (a tyrosine kinase), is degraded following exposure of T cells to activated granulocytes. Schmielau & Finn Cancer Research (2001) 61: 4765-4760; Malmberg et al., The Journal of Immunology (2001) 167:2595-2601; and Cemerski et al., Eur. J. Immunol. (2003) 33:2178-2185. Cemerski et al. summarized the state-of-the-art in 2003 by pointing out (see Cemerski et al., Introduction) that reactive oxygen species were known to affect protein structure by inducing various side chain modifications on cysteines, tyrosines, methionines, prolines, etc., by forming protein cross-linkages, and by oxidising the protein backbone, resulting in protein fragmentation. Collectively, these observations indicate that T cell function in stored blood samples is likely to be impaired when measured in a CMI assay.